One of the greatest obstacles facing basic and applied molecular Anopheles gambiae research is the prohibitively low efficiency of transgenesis for this species. In the twenty years since first report of a stable germline transformation, only a handful of successful transgenic An. gambiae lines have been published. The dearth of tools for targeted gene knockouts and gene replacements in Anopheles gambiae is currently recognized as a critical research need. In other systems, Zinc-Finger Nucleases (ZFNs) are very efficient tools for targeted gene-disruption by inducing a double strand break (DSB). When no homologous donor template is provided, ZFN-induced DSBs result high efficiency gene-disruption through non-homologous end-joining (NHEJ)-mediated frame-shifts. When a DSB occurs, if a homologous template (such as a plasmid) is available the DSB can be repaired by the homologous recombination (HR) pathway, inserting new genetic material into the genome. It has been shown in other systems that when a single-stranded parvovirus genome is used as the homologous template with a DSB, site-specific gene replacement occurs at a frequency of 10-1 to 10-3 events per cell - up to 10 million times greater efficiency than spontaneous HR (no DSB) with a traditional plasmid template. Our laboratory possesses the only known parvovirus capable of infecting An. gambiae (the Anopheles gambiae densonucleosis virus;AgDNV), which we believe will serve as a high-efficiency vector for the delivery of agents capable of precise manipulation of the Anopheles gambiae genome. In this proposal, we will investigate this question using an in vitro system. Our overall hypothesis is that AgDNV can be used to transduce specific ZFNs alone or in conjunction with homologous gene replacement cassettes for high- efficiency gene knockout or gene replacement. This hypothesis will be examined by the following 3 specific Aims: 1) establish a system for targeted gene-disruption by AgDNV-based transduction of site-specific ZFNs in Anopheles gambiae cells, 2) use recombinant AgDNV donor vectors for gene replacement by homologous recombination in Anopheles gambiae cells and 3) examine the synergistic effect of ZFN-mediated site-specific DSBs on AgDNV-mediated gene-replacement. PUBLIC HEALTH RELEVANCE: One of the greatest obstacles facing basic and applied molecular research on the major malaria vector Anopheles gambiae is the prohibitively low efficiency of transgenesis for this species. The Anopheles research community will benefit tremendously from tools that enable targeted gene knock-outs and targeted integration of transgenes. In this proposal, we will develop a system for ultra- high efficiency site-specific gene knock-outs and knock-ins for the Anopheles gambiae genome.